Labeling biological structures in cells has long been an important and challenging area of research, and many commercial fluorophores are available for making biological structures visible. Current interest in biological and medical fluorescence imaging of cells has pushed to the single-molecule regime, wherein the light from just one molecule can be detected. This technique provides a window into, for example, the operation of the various nanomachines inside cells as they undergo mechanochemical and enzymatic reactions. Single-molecule imaging then provides the opportunity to understand how these individual machines work and/or to assess if there is any pathology present on a molecular level. A number of diseases depend upon small numbers of errant molecules, so the ability to detect cellular processes all the way down to single molecules is a key area for development.
In addition, there is much interest in photoswitchable molecules, so that the individual emitters can be turned on, one at a time or a few at a time, in order to image the position of each single molecule below the optical diffraction limit. The optical diffraction limit is approximately the optical wavelength employed divided by two, which would be, for example, about 250 nm for 500-nm optical wavelength. This effect typically limits the resolution of optical microscopy so that two objects closer together than the optical diffraction limit cannot be resolved by conventional optical microscopy. Recent advances in optical imaging beyond the diffraction limit with single molecules (Betzig, E. et al., Science, 2006, 313, pp. 1642-1645; Hess, S. T., et al., Biophys. J., 2006, 91, pp 4258-4272; and Rust, M., et al., Nature Meth., 2006, 3, pp. 793-795) have overcome this limitation of resolution, but at the same time have introduced a new requirement for fluorescent labels that can be turned on at will: fluorophores must be actively controlled (usually via photoswitching or photoactivation) to ensure that only one single emitter is switched on at a time in a diffraction-limited region (Moerner, W. E., Proc. Nat. Acad. Sci. (USA), 2007, 104, pp. 12596-12602; Heileman, M. Laser & Photonics Reviews, 2009, 3, pp. 180-202; and Fernandez-Suarez, M, Ting, A. Y., Nature Reviews Molecular Cell Biology, 2008, 9, pp 929-943). The location of each of these sparse molecules is precisely determined beyond the diffraction limit, and a super-resolution image is obtained from the summation of the positions of single molecules from successive rounds of photoactivation. The ultimate spatial resolution is determined by a number of factors, most importantly the total number of photons detected from each single molecule (Thompson, R. E., et al., Biophys. J., 2002, 82, pp 2775-2783) and the density of the fluorogenic labels. Super-resolution imaging by these methods often uses photoactivatable fluorescent proteins, which have the advantage of being genetically targeted (Ando, R, et al., Science, 2004, 306, pp. 1370-1373; and Patterson, G. H., et al., Science, 2002, 297, pp 1873-1877); however, fluorescent proteins typically provide 10-fold fewer photons before photobleaching than good small-molecule emitters (Harms, G. S., et al., Biophys. J. 2001, 80, pp. 2396-2408). Therefore, there is a need to develop new photoactivatable organic fluorophores as well as methods to target said probes to specific locations or biomolecules in living cells. Examples of state-of-the art chemistries, photophysics, and targeting schemes include the following: Chen, I., et al, Curr. Opin. Biotech., 2005, 16, pp. 35-40; Prescher, J. A., et al., Nat. Chem. Biol., 2005, 1, pp. 13-21; Adams, S. R., et al., J. Am. Chem., Soc., 2002, 124, pp. 6063-6076; Bates, M., et al., Phys. Rev. Lett., 2005, 94, pp. 108101-1-108101-4; Folling, J., et al., Chem. Int. Ed., 2007, 46, 6266-6270; and Lord, S. J., et. al., 2008, J. Amer. Chem. Soc., 130, pp. 9204-9205.
Photoactivatable and chemically fluorogenic emitters have other important applications, including but not limited to photoaffinity labeling (PAL), spatial and temporal activation for diffusion studies, and mitigation of background fluorescence. For instance, one critical issue for the practical detection of fluorescence from single molecules in cells is the need to reduce the background emission signal that is due to the presence of extraneous and unwanted emitters. For example, some current cell-labeling schemes require incubation of a cell with a fluorophore, which may find a specific location in the cell by virtue of a special targeting moiety; however, those fluorescent molecules that are not correctly targeted must still be removed from the cell as their fluorescence may interfere with observation of the targeted fluorophore. This requires extensive washing of the cell to remove superfluous fluorescent tags. This washing step is also problematic in that it delays the observation of the molecules of interest until the washout is complete, hence dynamic phenomena are more difficult to observe. Worse, for the observation of single molecules, if the washout is not effective in removing all the superfluous fluorophores, the properly targeted single molecules are more difficult to distinguish and observe. It is desired to have bright organic fluorophores that can be turned on and/or off, and the ability to optically generate fluorophores in a specific region of a cell would reduce washout problems. Moreover, dark molecules that become fluorescent only after a bioconjugation chemical reaction also reduce the washing requirement.
Azide-based fluorogens have been reported previously, but they require short activation wavelengths, are not photostable enough to be applied to single-molecule imaging, react via a different mechanism, and do not produce fluorophores of the present invention (Dockter, M. E., J Biol. Chem., 1979, 254, pp. 2161-2164, and Dreyfuss, G., et al., Proceedings of the National Academy of Sciences, 1978, 75, pp. 1199-1203). The Dreyfuss system brightens not by a chemical or photochemical reaction, but due to confinement of the heterocycle in the cyclic adenosine monophosphate. The Dockter system utilized a fluorogenic photoactivatable azide that was a naphthalene-based molecule 3-azido-(2,7)-naphthalene disulfonate instead of a push-pull, donor-pi-acceptor system as described herein. In the Dockter report, the fluorescence is quenched by the n→pi* transition of the azide, instead of by disruption of a push-pull system, as in the current invention. Evidence of the different type of photoactivation can be found in the reference Moreland, et al. Anal. Biochem. 1980, 103, 26-32, which demonstrates that the azido compound “ANDS” absorbs at wavelengths red-shifted compared to the resulting fluorophore 3-amino-(2,7)-naphthalene disulfonate, while for the current invention the azido fluorogens are blue-shifted relative to the generated fluorophore. While this mechanism of quenching by the azide electronic transition may be applied to the specific naphthalene case in the Dockter report, it does not describe the current invention. The current invention applies to push-pull chromophore systems, and the mechanism of switching involves a creation of the fully conjugated donor-pi-acceptor system in the fluorophore by installation of one or more of the critical components that is absent in the fluorogen. This different mechanism is manifested by a dramatic red-shift of the absorption found in the product amine fluorophores compared to the initial azide fluorogens (as in FIG. 2b): by changing the azide group to an amine and pumping at the longer absorption wavelength of the amine, the final fluorophore compound now possess all three of the necessary components of the push-pull system and lights up. A fluorogenic PAL based on an azido coumarin (SAED) has also been reported (B. Thevenin, et al., Eur. J. Biochem. 1992, 206, 471-7), but in this and all the earlier cases in the literature, the light required to excite fluorescence is in the near ultraviolet (e.g. 280 and 334 nm), which prohibits ultrasensitive detection in living cells, because these short wavelengths additionally pump cellular autofluorescence and cause cell damage. Photoconversion in these previous cases was not accompanied by a significant spectral shift, and the subsequent fluorophores were not photostable enough to be applied to single-molecule imaging. Therefore, the current invention represents a dramatic improvement over the prior art.
In summary, the prior art was not sufficient for many cell-imaging applications, because the photogenerated fluorophores were not sufficiently bright, long-lived, or possessed red enough absorption. The novel azide fluorogens based on push-pull chromophores described in the present invention are a major improvement over the prior art because in the new system the absorption and emission of the donor substituted fluorophore obtained from the original azide substituted fluorogen is red shifted from that of the original fluorogen hence allowing for single-molecule imaging in living cells. Furthermore, the photogeneration wavelengths producing this change are closer to the visible regime. Because push-pull chromophores can be tuned over a range of wavelengths including the longer wavelengths, it is possible to design fluorogenic photoactivatable azido-fluorogens that are better suited to experiments involving living cells. Such fluorogens when converted to fluorophores are bright and emit millions of photons before photobleaching, and so are powerful tools for a variety of experiments requiring ultrasensitive detection of individual fluorescent molecules.